Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, University of Kansas Specialized Chemistry Center, University of Kansas, 2034 Becker Dr., Lawrence, KS 66047 and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
Nucleic Acids Research (Impact Factor: 9.11). 06/2012; 40(17):8607-21. DOI: 10.1093/nar/gks623
Source: PubMed
ABSTRACT
Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few
hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative.
After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin
Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds
that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50 = 1.4 μM), suramin sodium salt (IC50 = 3.6 μM), NF 023 hydrate (IC50 = 6.2 μM) and tyrphostin AG 538 (IC50 = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic
HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However,
when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of
the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific
for HCV NS3h than similarly potent HCV helicase inhibitors.

Full-text

Available from: David N Frick
Identification and analysis of hepatitis C virus NS3
helicase inhibitors using nucleic acid binding assays
Sourav Mukherjee
1
, Alicia M. Hanson
1
, William R. Shadrick
1
, Jean Ndjomou
1
, Noreena
L. Sweeney
1
, John J. Hernandez
1
, Diana Bartczak
1
, Kelin Li
2
, Kevin J. Frankowski
2
,
Julie A. Heck
3
, Leggy A. Arnold
1
, Frank J. Schoenen
2
and David N. Frick
1,
*
1
Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211,
2
University of Kansas Specialized Chemistry Center, University of Kansas, 2034 Becker Dr., Lawrence,
KS 66047 and
3
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla,
NY 10595, USA
Received March 26, 2012; Revised May 30, 2012; Accepted June 4, 2012
ABSTRACT
Typical assays used to discover and analyze small
molecules that inhibit the hepatitis C virus (HCV)
NS3 helicase yield few hits and are often con-
founded by compound interference. Oligonucleotide
binding assays are examined here as an alternative.
After comparing fluorescence polarization (FP),
homogeneous time-resolved fluorescence (HTRF
Õ
;
Cisbio) and AlphaScreen
Õ
(Perkin Elmer) assays,
an FP-based assay was chosen to screen Sigma’s
Library of Pharmacologically Active Compounds
(LOPAC) for compounds that inhibit NS3-DNA
complex formation. Four LOPAC compounds in-
hibited the FP-based assay: aurintricarboxylic acid
(ATA) (IC
50
= 1.4 kM), suramin sodium salt (IC
50
=
3.6 kM), NF 023 hydrate (IC
50
= 6.2 kM) and tyrphostin
AG 538 (IC
50
= 3.6 kM). All but AG 538 inhibited
helicase-catalyzed strand separation, and all but
NF 023 inhibited replication of subgenomic HCV rep-
licons. A counterscreen using Escherichia coli
single-stranded DNA binding protein (SSB) revealed
that none of the new HCV helicase inhibitors were
specific for NS3h. However, when the SSB-based
assay was used to analyze derivatives of another
non-specific helicase inhibitor, the main component
of the dye primuline, it revealed that some primuline
derivatives (e.g. PubChem CID50930730) are up to
30-fold more specific for HCV NS3h than similarly
potent HCV helicase inhibitors.
INTRODUCTION
All cells and viruses need helicases to read, replicate and
repair their genomes. Cellular organisms encode
numerous specialized helicases that unwind DNA, RNA
or displace nucleic acid binding proteins in reactions
fuelled by ATP hydrolysis. Small molecules that inhibit
helicases would therefore be valuable as molecular
probes to understand the biological role of a particular
helicase, or as antibiotic or antiviral drugs (1,2). For
example, several compounds that inhibit a helicase
encoded by herpes simplex virus (HSV) are potent drugs
in animal models (3,4). Despite this clear need, relatively
few specific helicase inhibitors have been reported, and the
mechanisms through which the most potent compounds
exert their action are still not clear. Although HSV
helicase inhibitors have progressed furthest in pre-clinical
trials (5), the viral helicase that has been most widely
studied as a drug target is the one encoded by the hepatitis
C virus (HCV). The uniquely promiscuous HCV helicase
unwinds duplex DNA and RNA in a reaction fuelled by
virtually any nucleoside triphosphate (6). The ability of
HCV helicase to act on DNA is particularly intriguing
because the HCV genome and replication cycle are
entirely RNA-based. There is no convincing evidence
that HCV helicase ever encounters DNA in host cells.
Compounds that disrupt the interaction of the helicase
and DNA, therefore, would be useful to understand why
an RNA virus encodes a helicase that acts on DNA. They
also might be useful antivirals because HCV needs a func-
tional helicase to replicate in cells (7) and helicase inhibi-
tors halt HCV replication in cells (8).
*To whom correspondence should be addressed. Tel: +1 414 229 6670; Fax: +1 414 229 5530; Email: frickd@uwm.edu
Present address:
Julie A. Heck, Department of Biology, College of Wooster, Wooster, OH 44691, USA.
Published online 27 June 2012 Nucleic Acids Research, 2012, Vol. 40, No. 17 8607–8621
doi:10.1093/nar/gks623
ß The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Page 1
The HCV helicase resides in the C-terminal two-thirds of
the viral multifunctional non-structural protein 3 (NS3),
which is also a protease. The NS3 protease and helicase
are covalently associated during HCV replication for
unknown reasons. HCV and related viruses encode the
only proteins known that are both proteases and helicases.
Recombinant DNA technology can be used to separate the
two NS3 functional domains, and express the proteins sep-
arately in Escherichia coli or other model organisms. Both
mono-functional, recombinant, truncated NS3 proteins
(called NS3p and NS3h) retain their activities in vitro.
Although NS3h retains a helicase function, its ability to
unwind RNA is somewhat diminished (9). The NS3
helicase was one of the first HCV enzymes to be charac-
terized, and crystal structures of NS3h were first solved in
the mid 1990s (10,11). However, helicase inhibitor develop-
ment has been far slower than it has been for other HCV
drug targets (2,12). To date, only a few classes of helicase
inhibitors have been reported to slow HCV RNA replica-
tion in cells. HCV helicase inhibitors reported to act as anti-
virals include nucleoside mimics (13), triphenylmethanes
(14), acridones (8,15), amidinoanthracyclines (16), tropo-
lones (17), symmetrical benzimidazoles (18–20) and pri-
muline derivatives (21).
One reason that so few molecular probes targeting HCV
helicase are available is because high throughput screens
for helicase inhibitors yield few hits. For example, a sen-
sitive molecular beacon-based helicase assay (MBHA) (22)
has been used to screen 290 735 compounds in the
NIH Molecular Libraries Small Molecule Repository
by the Scripps Research Institute Molecular Screening
Center (PubChem Project: http://pubchem.ncbi.nlm.nih.
gov/assay/assay.cgi?aid=1800, 14 June 2011, date last
accessed), and only 500 compounds (0.2%) were con-
firmed as hits upon retesting (PubChem Project: http://
pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1943, 14
June 2011, date last accessed). The most potent hits in
the NIH screen did not, however, directly inhibit
helicase action, but instead they interfered with the assay
(PubChem Project: http://pubchem.ncbi.nlm.nih.gov/
assay/assay.cgi?aid=485301, 14 June 2011, date last
accessed). They also did not inhibit HCV replication in
cells (PubChem Project: http://pubchem.ncbi.nlm.nih.
gov/assay/assay.cgi?aid=463235, 14 June 2011, date last
accessed) (23).
Screens for helicase inhibitors typically rely on assays
that monitor either helicase catalyzed strand separation or
ATP hydrolysis. Both assays are relatively complex, and
inhibitory compounds might act through the enzyme,
ATP, nucleic acid or other required cofactors (24). Both
assays monitor a helicase’s motor action, and it is possible
that the protein conformational changes that take place in
these assays, or some other unknown factor, obfuscates
inhibitor identification in large screens. Here, we test
whether simpler DNA-binding assays might be more
useful for HCV helicase inhibitor discovery.
Hepatitis C virus helicase binds single stranded DNA
and RNA with similar high affinities in the absence of
ATP. When ATP is present, it fuels helicase movements
and the subsequent separation of both DNA and RNA
duplexes (25). The ability of HCV helicase to separate
both DNA and RNA is intriguing because other similar
enzymes typically prefer DNA or RNA and because NS3
likely never encounters DNA. The RNA virus replicates in
the cytoplasm and has no DNA stage in its replication
cycle. Numerous NS3 crystal structures show how the
protein binds DNA (26–28) or RNA (29), both in the
absence or presence of ATP analogs (28,29). These struc-
tural studies reveal that amino acid side chains in the
NS3h nucleic acid binding cleft do not directly contact
the 2
0
-hydroxyl of RNA, explaining the enzyme’s
unusual promiscuity, and justifying the use of DNA oligo-
nucleotides as surrogates for RNA to probe the enzyme’s
functions. HCV helicase binds nucleic acids with low
nanomolar affinity (30,31) and NS3h preferentially inter-
acts with polypyrimidine tracts like those found in the 3
0
untranslated region of the virus genome (10,32). While it is
clear that one strand of DNA (or RNA) binds in a cleft
separating the two conserved helicase motor domains
from a third helical domain, it is not clear where else on
the protein nucleic acids might bind. Based on modeling
studies, some groups have suggested that RNA might bind
in the positively charged cleft separating the protease from
the helicase (12,33), and more recent evidence suggests the
protease region binds certain sequences in the internal
ribosome entry site of the HCV RNA genome (34).
We show here how DNA binding assays can be used
to identify new helicase inhibitors and how DNA binding
assays with unrelated proteins can be used to screen a
library of helicase inhibitors for specific compounds. A
truncated NS3 lacking the protease domain (i.e. NS3h)
is used because it is still unclear exactly how the
protease region affects NS3–RNA interactions, and
DNA is used here instead of more costly RNA, because
the nucleic acid-binding site on NS3h does not differenti-
ate between the DNA and RNA. First, we compare
various DNA binding assays for their screening utility.
Next, we use a fluorescence-polarization (FP)-based
binding assay to identify three new HCV helicase
inhibitors. Binding assays with the unrelated E. coli
single-stranded DNA binding protein (SSB) are then
used to reveal that the new compounds, like helicase in-
hibitors discovered in a prior screen of the NCI
Mechanistic Set (21), are not specific for HCV helicase.
In the final part of this study, we use a library of com-
pounds derived from a scaffold identified in the prior
screen (21) to show that binding assays can be used to
differentiate specific inhibitors from non-specific HCV
helicase inhibitors.
MATERIALS AND METHODS
Materials
DNA oligonucleotides were obtained from Integrated
DNA Technologies (Coralville, IA). HCV NS3h was
expressed and purified as described (6). Helicase sub-
strates were prepared by combining DNA oligonucleo-
tides (Integrated DNA Technologies, Coralville, IA) at a
1:1 molar ratio to a concentration of 20 mMin10mM
Tris–HCl pH 8.5, placing in 95
C water, and allowing
them to cool to room temperature for 1 h. The partially
8608 Nucleic Acids Research, 2012, Vol. 40, No. 17
Page 2
duplex helicase substrates possessing a 3
0
ssDNA tail
were then purified of free oligonucleotides by mixing
DNA 6:1 with 6X loading buffer (0.25% bromophenol
blue, 0.25% xylene cyanol FF, 40% sucrose) and sepa-
rating with 20% non-denaturing PAGE at a constant
200 V for 1 h.
Electrophoretic mobility shift assay
Binding assays containing 50 mM Tris, pH 7.4, 10%
glycerol, 100 nM DNA substrate (5
0
-Cy5-CC TAC GCC
ACC AGC TCC GTA GG–3
0
annealed to 5
0
-GGA GCT
GGT GGC GTA GG (T)20-3
0
) and 650 nM NS3h were
incubated 20 min on ice. Following addition of indicated
concentrations of thioflavine S, the binding reactions were
incubated another 20 min on ice. A BioRad precast 15%
polyacrylamide Tris/Borate/EDTA gel was pre-run at 4
C
for 30 min at 120 V. Four microliters of each sample was
loaded onto the gel. The gel was run 1 min at 200 V to
allow samples to enter gel, then 40 min at 120 V. The gel
was scanned on a Molecular Dynamics Storm 860
Phosphorimager.
FP-based DNA-binding assay
For screening, assays were performed in a total volume of
20.2 ml in 384-well, flat-bottom, low volume, black micro-
plates (Greiner Bio-One, catalog #784076). First, 20 mlofa
FP-assay solution (5 nM Cy5-TTTTTTTTTTTTTTT-3
0
(Cy5-dT15), 15 nM NS3h, 25 mM MOPS, pH 7.5,
1.25 mM MgCl
2
, 0.0025 mg/ml BSA, 0.005% (v/v)
Tween20 and 0.025 mM DTT) was dispensed in each
well, then 0.2 ml of dimethylsulfoxide (DMSO) or
compound dissolved in DMSO was added by pin
transfer, such that the final concentration of DMSO was
1% (v/v) in each assay.
For confirmation and IC
50
value determination, assays
were performed in half area 96-well microplates (Corning
Life Sciences, catalog #3694). First, 47.5 ml of a FP-assay
solution was dispensed in each well, then 2.5 ml of DMSO
or compound dissolved in DMSO was added, such that
the final concentrations in each assay were 5 nM Cy5-
dT15, 15 nM NS3h, 25 mM MOPS, pH 7.5, 1.25 mM
MgCl
2
, 0.0025 mg/ml BSA, 0.005% (v/v) Tween20,
0.025 mM DTT and 5% DMSO (v/v).
Polarization was monitored with a TECAN Infinite
M1000 PRO multi-mode microplate reader by exciting
at 635 nm (5 nm bandwidth) and measuring total fluores-
cence intensity, parallel and perpendicular polarized light
at 667 nm (20 nm bandwidth). G-factors were calculated
from wells with Cy5-dT15 alone. Inhibition (%) was
calculated by normalizing data to values obtained with
positive controls (200 nM dT20 or 100 mM primuline)
and negative controls (DMSO only). Assay interference
was calculated by dividing fluorescence intensity of a
compound-containing assay (F
c
) by the average fluores-
cence intensity of the negative controls (F
()
). Similar
results were obtained with both assay formats as long as
DMSO concentrations remained below 5%.
Compounds in the HCV helicase inhibitor library were
either purchased (Sigma, St. Louis, MO) or synthesized as
described (21) and screened at 20 mM. Samples in Sigma’s
Library of Pharmaceutically Active Compounds
(LOPAC) were screened at 100 mM.
Homogeneous time resolved fluorescence (HTRF
Õ
) assay
Assays were performed in 20.2 ml in 384-well, flat-bottom,
small volume, white microplates (Greiner Bio-One,
catalog #784075). The procedure was the same as that
for the FP assay except that 15 ml of the reaction
mixtures (7.5 nM Cy5-dT15 DNA, 50 nM NS3h, 25 mM
Tris, pH 7.5, 1.25 mM MgCl
2
, 0.05 mg/ml BSA, 0.1%
(v/v) Tween20, and 0.5 mM DTT) was first dispensed in
each well before the addition of 0.2 ml of dT20 or H
2
O.
After addition, 5 ml of a (1:50 dilution) of Lumi4
Õ
-Tb
Cryptate-conjugated anti-6 Histidine mouse monoclonal
antibody (catalog #61HISTLA, CISBIO US) was added
and the plate was incubated for 60 min at 4
C. TR-FRET
was monitored with a Fluostar Omega multimodal plate
reader (BMG Labtech, Inc.) by excitation of the donor
fluorophore at 340 nm. TR-FRET ratio was calculated
as emission of acceptor fluorophore at 665 nm over the
emission of donor fluorophore at 620 nm (gain 2300, inte-
gration time 400 ms, integration start time 60 ms, position-
ing delay 0.2 s, measurement start time 0 s, number of
flashes per well 200).
AlphaScreen
Õ
assay
All assays were similar to the above assays except
that they contained a final concentration of 10 nM
biotinylated oligonucleotide (Bio-d18, 5
0
-Bio-GCC TCG
CTG CCG TCG CCA-3
0
), instead of Cy5-dT15, and
they used reagents from the AlphaScreen
Õ
Histidine
(Nickel Chelate) Detection Kit (catalog #6760619C,
Perkin Elmer). To 384-well, flat-bottom, low volume,
white microplates (Greiner Bio-One, catalog #784075),
12 ml of the reaction mixtures containing 10 nM Bio-d18,
20 nM NS3h, 25 mM HEPES, pH 7.5, 100 mM NaCl and
1.0 mg/ml BSA was dispensed, followed by 0.2 ml of dT20
or H
2
O and 4 ml Anti-His alpha screen donor beads. After
incubation for 30 min at 23
C, 4 ml of streptavidin-
acceptor beads was added, and the assays incubated
another 60 min. All work with the alpha reagents was per-
formed under green filtered light conditions (<100 Lux).
Alpha counts were measured at 520–620 nm (Ex. 680 nm,
20 nm bandwidth) in a Fluostar Omega multimodal plate
reader (BMG Labtech, Inc.).
NS3h MBHAs
The ability of compounds to inhibit helicase action was
monitored using molecular beacons as described previously
(19,22). Assays contained 25 mM MOPS, 1.25 mM MgCl
2
,
5% DMSO, 5 mg/ml BSA, 0.01% (v/v) Tween20, 0.05 mM
DTT, 5 nM substrate, 12.5 nM NS3h and 1 mM ATP. The
partially duplex DNA substrates used in MBHAs consisted
of a 45-mer bottom strand 5
0
-GCT CCC CGT TCA TCG
ATT GGG GAG C(T)20-3
0
and the 25-mer HCV top
strand 5-/5Cy5/GCT CCC CAA TCG ATG AAC GGG
GAG C/3IAbRQSp/-3. The 3-stranded RNA substrate
used was made of two RNA strands, a 60 nucleotide
long bottom strand 5
0
-rGrGrA rGrCrU rGrGrU rGrGrC
rGrUrA rGrGrC rArArG rArGrU rGrCrC rUrUrG
Nucleic Acids Research, 2012, Vol. 40, No. 17 8609
Page 3
rArCrG rArUrA rCrArG rCrUrU rUrUrU rUrUrU
rUrUrU rUrUrU rUrUrU rUrUrU-3
0
, a 24 nucleotide
long top strand 5
0
-rArGrU rGrCrG rCrUrG rUrArU
rCrGrU rCrArA rGrGrC rArCrU-Cy5, and a
third DNA top strand with the sequence/5IAbRQ/CCT
ACG CCA CCA GCT CCG TAG G-3. In the screen
in Figure 5, percent inhibition was calculated with
equation (1) and interference with equation (2).
Inhibition %ðÞ¼ððFc
0
=Fc
30
ÞðFðÞ
0
=FðÞ
30
Þ=
ð1 ðF ðÞ
0
=F ðÞ
30
ÞÞÞ 100
ð1Þ
Interference ratioðÞ¼ðFc
0
=FðÞ
0
Þð2Þ
In equations (1) and (2), Fc
0
is the fluorescence of the
reactions containing the test compound before adding
ATP, Fc
30
is the fluorescence of the test compound
reaction 30 min after adding ATP. F()
0
is the average
of three DMSO-only negative control reactions before
adding ATP and F()
30
is the average of three
DMSO-only reactions 30 min after adding ATP.
To monitor helicase reaction kinetics and to calculate
IC
50
values, assays were performed in a volume of 60 mlin
white half-area 96-well plates (Corning Lifesciences,
catalog #3693) and measured in a Fluostar Omega multi-
modal plate reader (BMG Labtech, Inc.) using the 640 nm
excitation wavelength and 680 emission wavelength filters.
Reactions were performed by first incubating all compo-
nents except for ATP for 2 min, then initiated by injecting
in 1/10 volume of ATP such that the final concentration of
all components was as noted above. Initial reaction
velocities were calculated by fitting a first order decay
equation to data obtained after ATP addition and
calculating an initial velocity from the resulting amplitude
and rate constant. The concentration at which a
compound causes a 50% reduction in reaction velocity
(IC
50
) was calculated using GraphPad Prism (v. 5).
HCV replicon assays
The ability of compounds to inhibit HCV replication was
judged using an HCV Renilla luciferase (HCV RLuc)
reporter construct that was a generous gift from
Seng-Lai Tan (35). Plasmid DNA expressing the
replicon was transcribed, and the subsequently purified
RNA was used to prepare stably transfected Huh7.5
cells by prolonged selection with G418. To test com-
pounds, HCV RLuc replicon containing cells were
seeded at a density of 10 000 cells per well in 96-well
plates and incubated for 4–5 h to allow the cells to
attach to the plate in 100 ml of DMEM supplemented
with 10% fetal bovine serum (HyClone), 2 mM
L-glutam-
ine, 100 U/ml penicillin, 100 mg/ml streptomycin and 1x
non-essential amino-acids (Invitrogen). To each well,
0.5 ml of compounds dissolved in DMSO were added
such that the DMSO final concentration was 0.5%,
and the cells were incubated for 72 h at 37
C under 5%
CO
2
atmosphere. The effect of compounds on HCV
replication was estimated by measuring the Renilla
luciferase activity using the Renilla luciferase assay
system (Promega, Madison, WI) in 96-well black
microplates (Thermo Scientific, catalog #9502867) read
on a FLUOstar Omega multi-mode microplate reader
(BMG Labtech, Inc.). Relative percent inhibition was
calculated by normalizing values to those obtained with
cells treated with DMSO only.
Cell viability assay
To assess compound toxicity towards Huh-7.5 cells, cells
were plated and treated as above and viability was
assessed using the Cell Titer-Glo luminescent cell viability
kit (Promega) following the manufacturer’s instructions.
Briefly, at the end of a 72 h incubation period, the medium
was removed and the cells were washed with growth
medium, then an equal volume of growth medium
and Cell Titer-Glo reagent was added and the lysis was
initiated by mixing on an orbital shaker. The plate
was incubated at 23
C for 30 min and luciferase activity
was measured for 1 s using a FLUOstar Omega microplate
reader (BMG Labtech, Inc.) in black 96-well microplates
(Thermo Scientific, catalog #9502867). Relative viability
was calculated by normalizing the values to those obtained
with cells treated with DMSO only.
Escherichia coli SSB assay
The procedure for screening with this assay was the same
as that for the FP-based DNA binding assay carried out in
384-well plates except that E. coli SSB (Promega) was used
at 20 nM instead of the HCV helicase. For IC
50
determin-
ation, assays were performed with 60 ml total volume in
black flat bottomed 384-well microplates (Corning catalog
#3573). First, 3.0 ml of DMSO or compound dissolved in
DMSO was added, such that the final concentration of
DMSO was 5% (v/v) in each assay. Then 57 mlofa
FP-assay solution (5 nM Cy5-dT15, 20 nM SSB, 25 mM
MOPS, pH 7.5, 1.25 mM MgCl
2
, 0.0025 mg/ml BSA,
0.005% (v/v) Tween20 and 0.025 mM DTT) was dispensed
in each well. Polarization was monitored as described
above.
RESULTS
Belon and Frick (36) previously reported that thioflavine S
was an HCV helicase inhibitor, and thioflavine S was used
as a positive control for the screen of the NIH Molecular
Libraries Small Molecule Repository (PubChem BioAssay
AID #1800) and in other studies (37). While studying
the mechanism of action of thioflavine S (Direct Yellow
7, Sigma Cat. #T1892) and the related yellow dyes
primuline (Direct Yellow 59, MP Biomedicals Cat.
#195454) and titan yellow (Direct Yellow 9/Thiazole
Yellow G, Sigma Cat. #88390) (38), we observed that
they prevent NS3h from binding its nucleic acid substrate
(Figure 1A). In the absence of one of these dyes, NS3h
binds its substrate tightly enough that the complex will
migrate more slowly through a non-denaturing polyacryl-
amide gel. When thioflavine S was present the gel-shift
of the substrate decreased in a concentration-dependent
fashion (Figure 1B).
8610 Nucleic Acids Research, 2012, Vol. 40, No. 17
Page 4
HCV helicase-DNA binding assays that are suitable for
high throughput screening
To unwind a substrate, HCV helicase binds a single
stranded nucleic acid tail then translocates in a 3
0
–5
0
dir-
ection (39) until it reaches a duplex region that it can
separate. The ability of NS3h to bind its substrate can
also be measured by monitoring changes in polarization
(or anisotropy) of a fluorescent helicase substrate as has
been done with related helicases (40,41). This loading step
can be monitored using a truncated substrate lacking
duplex regions, such as a 15-nucleotide long deoxythy-
midine polymer (dT15) (Figure 2A). Previous work has
shown that NS3h binds such single stranded DNA with
a high affinity and that 2-3 protomers bind such an oligo-
nucleotide. A homopolymer was chosen to minimize the
possibility that the DNA would form a hairpin or other
secondary structures, and Ts were chosen because NS3h
prefers this sequence to others (10,30–32).
As seen before (30,31,42), the binding of NS3 to DNA
in this FP-assay was stoichiometric (K
d
< 1 nM) and
about two to three molecules of NS3 were needed to
saturate Cy5-dT15. When 5 nM Cy5-dT15 was present,
increasing amounts of NS3h increased the FP of
Cy5-dT15 in a concentration dependent manner such
that the amount of NS3h needed to bind half of the
Cy5-dT15 (K
0.5
) was 9 ± 1.5 nM. Under these conditions,
the signal plateaued when about three times as much
NS3h (15 nM) was added as the amount of Cy5-dT15
(Figure 2B). The polarization of a Cy5-dT15–NS3h
complex decreased in the presence of either unlabeled
ligand (dT20) or a yellow dye in a concentration depend-
ent manner (Figure 2C). The IC
50
values measured for
dT20 and primuline were 7 ± 2 nM and 24 ± 3 mM, re-
spectively. Thioflavine S decreased polarization less effect-
ively than primuline with an IC
50
of 35 ± 3.5 mM, and
titan yellow was 10 times more potent than either with
an IC
50
value of 2.8 ± 0.2 mM (Table 1).
A Cy5-labeled oligonucleotide was chosen mainly
because its fluorescence intensity did not change upon
protein binding, and because it absorbs and emits light
in the far-red visible range, where it would be less likely
to interact with compounds in large chemical libraries.
One possible problem with using Cy5 as a tracer in such
a study is that it is coupled to the oligonucleotide with an
aliphatic linker so that the fluorophore could, in theory, be
still relatively free to rotate even when DNA is bound to
the enzyme, a phenomenon commonly referred to as ‘the
propeller effect’. We therefore also tested oligonucleotides
labeled with fluorescent moieties not bound to aliphatic
linkers such as with 6-carboxyfluorescein, hexachloro-
fluorescein and boron-dipyrromethene (BODIPY).
Polarization studies with each of these alternatives were
confounded by the fact that the fluorescence intensity of
each changed upon protein binding. The fact that fluores-
cence intensity of oligonucleotides changes when they bind
the unusually acidic DNA binding site of NS3 has been
previously documented (9,19). While screening fluorescent
oligonucleotides, we found two other red-shifted tracers
that did not change intensity upon binding, Tye
TM
665
and Alexa Fluor 647
TM
. Identical (dT15) oligonucleotides
labeled on the 5
0
-end with either Cy5, Tye665, or Alexa
Fluor 647, bound NS3h with similar K
0.5
s (5 ± 1, 8 ± 2
and 6 ± 2, respectively), and titan yellow inhibited
complex formation of each with a similar IC
50
value
(7 ± 3, 5 ± 1 and 9 ± 4 mM, respectively). Repeated
assays (n = 40) with the three different fluorescence
tracers in the presence and absence of titan yellow
(100 mM) revealed similar Z
0
factors (43). Assays with
Alexa Fluor and Tye 665 labeled oligonucleotides had
the largest difference between the positive and negative
controls, but their assay-to-assay variability was higher,
particularly in assays done in the absence of inhibitor
(Figure 2D). Further experiments were therefore per-
formed with the Cy5-labeled oligonucleotide.
After optimizing conditions, FP-based binding assays
were then performed in a high throughput format to
judge necessary precision and reproducibility. To judge
well-to-well variation, 48 negative controls (DMSO only)
and 48 positive controls (100 mM primuline) were per-
formed. The coefficient of variation was 2.2% for the
negative controls and 5.8% for the positive controls, re-
sulting in a Z
0
factor of 0.81 (Figure 2E). Similar Z
0
factors, and concentration response curves were
obtained when plates were compared (Figure 2F) or
AB
Figure 1. Thioflavine S inhibits the ability of NS3h to bind to its DNA substrate. (A) Partially duplex DNA helicase substrate used for gel-shift
analysis. (B) Electrophoretic mobility shift assay (EMSA). Samples containing the MBHA substrate (100 nM), NS3h (650 nM) and indicated con-
centrations of thioflavine S were examined on a 15% native polyacrylamide gel using a phosphorimager to locate labeled DNA. Control lane with no
NS3h shows migration position of free DNA.
Nucleic Acids Research, 2012, Vol. 40, No. 17 8611
Page 5
assays were performed on different days (Figure 2G). To
judge reproducibility, duplicate assays were performed
with 143 different HCV helicase inhibitors at a concentra-
tion of 20 mM. All but two samples in this library
decreased polarization of the Cy5-dT15–NS3h complex
to some extent in a reproducible fashion. Two compounds
in the collection, the DNA binding dyes H33258 and
TO-PRO3 (Invitrogen), increased polarization, suggesting
that they bound the Cy5–DNA–NS3h complex but did
not displace the oligonucleotide (Figure 2H).
Two other methods that are frequently used to monitor
protein nucleic acid interactions were compared with the
above FP-based assay. The first was a modified homoge-
neous time resolved fluorescence (HTRF
Õ
) assay (Cisbio
bioassays), in which Cy5-dT15 was used as an acceptor of
long-lived lanthanide fluorescence. While optimizing this
TR-FRET assay, we tested three different lanthanide
donors: a Lumi4
Õ
-Tb Cryptate-conjugated anti-6 Histidine
mouse monoclonal antibody (catalog #61HISTLA,
Cisbio), an Eu
3+
Cryptate-conjugated mouse anti-6
Histidine monoclonal antibody (catalog #61HISKLA,
Cisbio) and a LANCE
Õ
Europium Anti-6X Histidine
antibody (catalog #AD0110, Perkin Elmer). The highest
Z
0
factors were obtained with the Tb
3+
cryptate (data not
shown). The optimized TR-FRET-based assay was per-
formed in the same buffer as the FP-based assays, except
that diluted anti-6 His antibody was added to each assay.
In the HTRF
Õ
assay setup, the ratio of signals from the
donor and acceptor is multiplied by 10 000 to estimate
TR-FRET. In our assay, time resolved fluorescence
occurring after excitation at 340 nm was measured at
625 nm, and the signal resulting from binding was detected
at a wavelength of 665 nm due to energy transfer to the
Cy5 (Figure 3A). The maximal energy transfer resulted in a
3.5-fold increase of the signal ratio in the presence of NS3h
bound to the Tb
3+
-conjugated anti-hexahistidine antibody.
This signal change returned to baseline upon addition of
A
BC
E
F
G
H
D
Figure 2. Fluorescence polarization (FP)-based assay to monitor the
interaction of HCV helicase and a deoxythymidine polymer. (A)FP
based assay to monitor NS3h binding to Cy5-dT15. (B) Fluorescence
polarization of Cy5-dT15 (5 nM) at different concentrations of NS3h.
Data (n = 4) were fitted to a concentration-response equation (four
Figure 2. Continued
parameter, variable slope) with dotted lines showing the 95% confi-
dence intervals for the curve fit. (C) Concentration response of un-
labeled dT20 (squares) or primuline (circles) on the fluorescence
polarization of a Cy5-dT15-NS3h complex. Data (n = 4) were fitted
to a four-parameter concentration response equation (variable slope)
constrained to values obtained in the absence of inhibitor (top) and the
absence of NS3h (bottom), with indicated IC
50
values and Hill slopes.
(D) Comparison of results obtained with Cy5-dT15 with those obtained
with dT15 labeled with either Tye665 or AlexaFluor 647.
Oligonucleotides were present at 5 nM and NS3h at 15 nM. Positive
controls (N = 40) contained 100 mM titan yellow, negative control con-
tained DMSO only. (E) Fluorescence polarization of 48 positive control
assays (100 mM primuline (+)) and 48 negative control assays (DMSO
only ()). Solid lines represent means and dotted lines 3 times the
standard deviations of the mean of all assays. (F) Normalized percent
inhibition of Cy5-dT15 complex formation by various concentrations of
dT20 observed in FP-assays performed on two different plates.
(G) Normalized percent inhibition of Cy5-dT15 complex formation
by various concentrations of dT20 observed in FP-assays performed
on two different days. (H) Correlation plot of fluorescence polarization
values observed in duplicate assays at 20 mM of samples in an HCV
helicase inhibitor library (Table S1). Data were fitted to a straight line
through zero (slope = 0.97, R
2
= 0.99). The dotted lines show values
representing 0% and 100% inhibition, as determined from negative
controls (DMSO only) and positive control (100 mM primuline).
8612 Nucleic Acids Research, 2012, Vol. 40, No. 17
Page 6
unlabeled oligonucleotide, dT
20
(200 nM) (Figure 3B).
Compounds that disrupt the Cy5-dT15-NS3h complex
also decreased the TR-FRET signal in a concentration-
dependent fashion with IC
50
values similar to those
determined with the FP-based assay (data not shown),
but the Z
0
-factor (0.59) for the TR-FRET assay was less
than what was observed with FP-based assay (Figure 3B).
The second assay compared to the FP-based assay was
based on the AlphaScreen
Õ
Histidine (Nickel Chelate)
Detection Kit (catalog #6760619C, Perkin Elmer). This
AlphaScreen
Õ
assay monitored the binding of NS3h to
DNA, using donor beads containing Ni
2+
ions that
interact with the C-terminal His-tag of NS3h and
streptavidin-bound acceptor beads binding to biotinylated
oligonucleotide (Bio-d18) (Figure 3C). Formation of an
NS3h–DNA complex brings the donor and acceptor
close enough that singlet oxygen can be transferred from
the donor to acceptor beads. Compounds that disrupt the
Bio-d18–NS3h complex (e.g. dT20) decrease the
AlphaScreen
Õ
signal (Figure 3D). The signal/background
in this AlphaScreen
Õ
was better than was seen in other
assays, with the complex yielding 80 - to 100-fold
increased counts, but assay variability was higher than
with the FP assay, leading to a Z
0
factor of 0.62, which
was again less than what was observed in the FP-based
assay.
Compound interference and advantage
over unwinding assays
Most of the compounds in the above helicase inhibitor
library were previously shown to inhibit NS3h when
screened using an MBHA (21). The MBHA (22)
monitors the ability of a helicase to remove a molecular
beacon (44) bound to a complementary strand upon ATP
addition (Figure 4A). In the absence of inhibitors, fluor-
escence decreases in the MBHA upon ATP addition, but
when an inhibitor (e.g. titan yellow) is present, fluores-
cence decreases at a slower rate (Figure 4B). The main
Table 1. Effects of yellow dyes and small molecules on the interaction of NS3h with DNA, its ability to unwind DNA and RNA, and HCV rep-
lication in cells
Compound DNA binding assays
a
NS3h helicase assays
b
Cellular assays
NS3h IC
50
(mM) ± SD
SSB IC
50
(mM) ± SD
DNA IC
50
(mM) ± SD
RNA IC
50
(mM) ± SD
HCV replicon
c
IC
50
(mM) ± SD
Viability
c
CC
50
(mM) ± SD
Thioflavine S 35 ± 3.5 12 ± 0.9 24 ± 1.3 22 ± 6.3 >100 >100
Primuline 24 ± 3 5.0 ± 1.4 12 ± 1.3 15 ± 2.3 >100 >100
Titan yellow 2.8 ± 0.2 2.9 ± 0.2 12 ± 3.6 30 ± 3.6 >100 >100
ATA 1.4 ± 0.1 1.0 ± 0.1 0.6 ± 0.1 0.8 ± 0.2 98 ± 30 >100
AG 538 3.6 ± 0.2 1.2 ± 0.1 >100 >100 18 ± 3.2 60 ± 13
NF 023 6.3 ± 0.6 1.7 ± 0.2 2.6 ± 0.4 5.1 ± 1.2 >25
d
>25
d
Suramin 3.6 ± 0.3 0.3 ± 0.0 3.7 ± 0.7 8.9 ± 2.0 38 ± 9.3
e
>50
e
a
Average (±SD) IC
50
value from three sets of FP-based binding assay performed with a 16 point 1.5-fold dilution series of each compound starting
at 100 mM.
b
Average (±SD) IC
50
value from 3 sets of molecular beacon based helicase assays performed with a 16 point 1.5-fold dilution series of each
compound starting at 100 mM.
c
Average (±SD) IC
50
value from three sets of assays performed with a 8 point 2-fold dilution series starting at 100 mM.
d
Average (±SD) IC
50
value from three sets of assays performed with a 8 point 2-fold dilution series starting at 25 mM.
e
Average (±SD) IC
50
value from three sets of assays performed with a 8 point 2-fold dilution series starting at 50 mM.
A
B
C
D
Figure 3. HTRF
Õ
and AlphaScreen
Õ
assays that detect NS3h interactions with DNA. (A) TR-FRET assay using the Lumi4
Õ
-Tb
cryptate-conjugated anti-hexahistidine (Cisbio Bioassays) as a donor and Cy5-dT15 as an acceptor. (B) TR-FRET observed with 5 nM Cy5-dT15
alone, with 15 nM NS3h, and with 15 nM NS3h and 200 nM dT20. Error bars are standard deviations (n = 16). (C) Use of AlphaScreen
Õ
Histidine
(Nickel Chelate) Detection Kit (Perkin Elmer) reagents to monitor NS3h binding to a biotinylated oligonucleotide. (D) AlphaScreen
Õ
counts for
control assays containing 10 nM Bio-d18 alone, with 20 nM NS3h, and with NS3h and 200 nM dT20. Error bars are standard deviations (n = 16).
Nucleic Acids Research, 2012, Vol. 40, No. 17 8613
Page 7
Figure 4. Comparison of HCV helicase DNA binding and unwinding assays. (A) The molecular beacon helicase assay (MBHA). (B) Effect of
indicated concentrations of titan yellow on MBHAs. (C) Effect of indicated concentrations of thiazole orange on MBHAs. (B) and (C) show both the
fluorescence traces for each reaction and the curve fits used to determine initial rates of DNA unwinding. (D) Mean percentage inhibition (equation
(1)) and compound interference (equation (2)) of duplicate FP-based binding assays (+) performed with samples from a library of HCV helicase
inhibitors. (E) Mean percentage inhibition (equation (1)) and compound interference (equation (2)) of duplicate MBHAs performed with samples
from a library of HCV helicase inhibitors. In (D) and (E), the dotted line denotes 0% inhibition and the solid vertical line denotes no interference.
(F) Percentage inhibition seen in the MBHA and FP-based assay for DNA binding compounds present in the library screened in panels (D) and
(E). Note that most DNA-binding compounds that inhibited the MBHA screen did not inhibit the FP-binding assay. (G) Percentage inhibition seen
in the MBHA and FP-based assay for the primuline derivatives present in the library screened in panels (D) and (E). Line shows the correlation of
the ability to inhibit both unwinding and binding. Full data for both screens are in Table S1.
8614 Nucleic Acids Research, 2012, Vol. 40, No. 17
Page 8
problem with using the MBHA, or similar unwinding
assays to screen for helicase inhibitors, is that it is difficult
to distinguish helicase inhibitors from compounds that
simply interfere with the assay by binding DNA, such as
thiazole orange (45) (Figure 4C). In screens, the ability of
a compound to inhibit the MBHA is measured by
comparing the fluorescence of the MBHA substrate
before and after ATP addition. If a compound simply
reduces fluorescence of the Cy5 substrate before ATP is
added, as seen with concentrations of thiazole orange
>12.5 mM (Figure 4C), it might falsely appear to inhibit
the helicase. A simple method to identify true helicase
inhibitors is to plot percent inhibition vs. compound inter-
ference, which is calculated by dividing assay fluorescence
before ATP addition (F
0
) by the fluorescence seen in
negative control reactions (F
0()
), which lack any inhibi-
tory compounds (21). To compare the results of a DNA
binding assay with the MBHA, the same library of known
HCV helicase inhibitors, which was screened with the
FP-based binding assay (Figure 4D), was re-screened
with the MBHA at the same compound concentration
(20 mM) (Figure 4E). When each screen was analyzed for
both percent inhibition and compound interference, it was
clear that fewer compounds interfered with the FP-based
binding assay (Figure 4D) than with the MBHA
(Figure 4E). For full results, see Table S1 (Supplementary
Data). The fact that fewer compounds interfered with
the FP-based assay suggests that many library samples
did not decrease fluorescence by simply quenching Cy5
fluorescence.
Another way that a compound might decrease the fluor-
escence of the MBHA substrate would be to distort the
duplex region such that the quenching moiety of the
beacon is more likely to interact with the Cy5 fluorophore.
If that were the case, then most DNA binding compounds
should appear to inhibit the MBHA but not the FP-based
binding assay, which lacks a duplex region. To test this
hypothesis, the average percent inhibition observed with
each compound in FP-based binding assays was compared
with the average percent inhibition seen in the MBHA
unwinding assay. Such a plot reveals that most of the
known DNA binding compounds in our HCV helicase
inhibitor library (e.g. berenil, proflavin, netropsin and
SYBR green I) inhibit the MBHA but not the FP-based
binding assay (Figure 4F). In contrast, compounds, which
act mainly by inhibiting the ability of NS3h to bind DNA,
such as those derived from primuline (21), inhibit both the
FP-binding assay and MBHA with a similar potency
(Figure 4G).
Identification of NS3h inhibitors
in Sigma’s LOPAC 1280
TM
To test if a DNA binding assay could be used to identify
new HCV helicase inhibitors, the above FP-based assay
was used to screen Sigma’s 1280-compound LOPAC. All
LOPAC compounds were screened at 100 mM in 384-well
plates, each containing positive (primuline or dT
20
) and
negative (DMSO or buffer only) controls (Figure 5A). Out
of the 1280 samples screened (Table S2), 18 compounds
exhibited FP signals significantly different from other
library samples. To identify compounds that decreased
FP by quenching of the fluorescence signal or intrinsic
fluorescence, the fluorescence intensity of each compound
(I) was divided by the fluorescence intensity observed for
negative controls (I
()
). A plot of this data (I/I
()
) revealed
that four hits increased or decreased the fluorescence in-
tensity by more than 20% in comparison with the negative
controls. Four of the compounds with similar fluorescence
intensity to that of the negative controls decreased FP by
more than 60% at a concentration of 100 mM (Figure 5B).
Each of these compounds [ATA (Sigma Cat. #A1895),
suramin sodium salt (Sigma Cat. #S2671), NF 023
hydrate (Sigma Cat. #N8652) and tyrphostin AG 538
(Sigma Cat. #T7822)] decreased polarization in a
concentration-dependent manner (Figure 5C–F). The
IC
50
values with the four best inhibitors were similar to
those seen with titan yellow and lower than those seen
with primuline and thioflavine S (Table 1).
To test if compounds that decrease FP of the
Cy5-dT15–NS3h complex inhibit the ability of NS3h to
unwind DNA, various concentrations of each of the com-
pounds above were added to MBHAs that monitor either
DNA or RNA unwinding. Three of the four compounds
identified in the LOPAC screen inhibited DNA-based
MBHAs (Figure 6B). Those that inhibited the ability of
NS3h to unwind DNA also inhibited its activity on an
RNA substrate (Table 1).
To determine if compounds that interact with the
NS3h–DNA complex might also function as antiviral
agents, their ability to inhibit the replication of an HCV
subgenomic replicon was tested (46). HCV RNA replica-
tion was measured using a reporter system in which a
Renilla luciferase gene was fused to the 5
0
-end of the
neomycin phosphotransferase gene needed for replicon se-
lection (Figure 6C), so that the cellular levels of Renilla
luciferase correlated directly with the amount of HCV
RNA present in cells (35). After replicon transfection
and selection, cells were treated in parallel in two sets of
triplicates. One set of cells was used for Renilla luciferase
assays and the other set was used to determine cell viabil-
ity using a firefly luciferase-based assay. Three of the four
compounds identified in the LOPAC screen inhibited
replicon luciferase (Figure 6D), and all four compounds
showed little sign of toxicity at a concentration of 25 mM
(Figure 6E). Selectivity was estimated by comparing the
potency with which the compound inhibits the replicon to
its toxicity. By this measure, AG 538 was the most select-
ive because three times more of this compound was needed
to reduce viability than was needed to inhibit HCV repli-
cation (Table 1).
A similar FP-assay using the E. coli SSB
The specificity of the new HCV helicase inhibitors was
examined using a counterscreen in which NS3h was
substituted with the unrelated E. coli SSB (Figure 7A).
Like NS3h, SSB increased the FP of Cy5-dT15, but
more (20 nM) SSB was needed to saturate the oligo-
nucleotide with a K
0.5
of 9.6 ± 2 nM in the presence of
5 nM Cy5-dT15 (Figure 7B). All the compounds that in-
hibited the Cy5-dT15–NS3h interaction also inhibited the
Nucleic Acids Research, 2012, Vol. 40, No. 17 8615
Page 9
interaction between Cy5-dT15 and SSB (Figure 7C and
Table 1). The specificity of each compound was judged
by comparing the IC
50
values obtained with NS3h and
SSB. By this measure, none of the new HCV helicase in-
hibitors were more specific than the yellow dyes (Table 1).
The yellow dyes were therefore selected to probe if the
SSB based counter screen could be used to identify specific
HCV helicase inhibitors. Thioflavine S was discovered to
inhibit HCV helicase in an MBHA-based screen of the
NCI Mechanistic Set of compounds (Figure 8). Li et al.
(21) isolated eight compounds from thioflavine S and the
related dye primuline and used the core dimeric
benzothiazole scaffold found in all eight components to
synthesize a library of semi-synthetic primuline deriva-
tives. Since we showed above that primuline inhibits
NS3h from binding DNA in a non-specific manner, we
screened the entire primuline derivative collection for
compounds that might be more specific. To this end, we
compared the ability of the compounds synthesized from
the main component of primuline to disrupt the
Cy5-dT15–SSB complex with their ability to inhibit the
HCV helicase in a standard HCV helicase MBHA
(Figure 8 and Table S3). This structure activity relation-
ship reveals that small changes to this scaffold can affect
the affinity of a compound for HCV helicase relative to its
ability to inhibit SSB from binding DNA. The most potent
and specific compound in this family, CID50930730, is
over 30 times more specific (as judged by the ratio of
IC
50
values for each compound in the MBHA to its IC
50
value in SSB-DNA binding assays, for each compound)
than the least specific compound with similar potency in
the MBHA, CID49849276 (Figure 8 and Table S3).
DISCUSSION
This study shows how DNA-binding assays can be used to
discover and characterize small molecules that inhibit
helicases. The assays are simpler than those used to
monitor helicase-catalyzed DNA unwinding or ATP hy-
drolysis, making them more amenable to high throughput
screening (HTS). Despite its simplicity, the FP-based
DNA binding assay developed here was able to find four
compounds that disrupt the HCV helicase-DNA inter-
action, three of which also inhibit the NS3h’s ability to
unwind DNA and RNA. Three compounds also inhibit
replication of subgenomic HCV replicons. Similar binding
assays using unrelated protein SSB were shown to be
useful for judging compound specificity, as was demon-
strated both with the newly identified helicase inhibitors
and with a panel of compounds created in a prior SAR
study of the primuline scaffold (21).
A
B
C
E
D
F
Figure 5. Identification of inhibitors of Cy5-dT15-NS3h complex for-
mation in a screen of the Sigma LOPAC 1280
TM
.(A) Summary of
screening results of a fluorescence polarization assay to identify inhibi-
tors of the Cy5-dT15-NS3h interaction among the 1280 samples in
Sigma’s LOPAC (+). Positive controls contained primuline (squares)
or dT20 (circles) and negative controls contained DMSO only (not
shown). The solid line represents the mean of all assays (except
positive controls) and the dotted lines three standard deviations.
(B) Normalized inhibition (%) for compounds that fall outside the
above three standard deviation limit are plotted against the
compound interference, defined as fluorescence intensity divided by
the average fluorescence intensity of the negative control samples.
The vertical dotted lines denote defined boundaries of tolerance for
either possible quenching (I = 0.8) or possible intrinsic fluorescence
(I = 1.2). The horizontal dotted line denotes arbitrary cut-off criterion
of 60% inhibition. Fluorescence polarization of a Cy5-dT15-NS3h
Figure 5. Continued
complex in the presence of increasing concentrations of (C) ATA,
(D) AG 538, (E) NF 023, or (F) Suramin. Data (n = 3) were fitted to
4-parameter concentration response curves constrained to values
obtained in the absence of inhibitor (top) and the absence of NS3h
(bottom) with parameters in Table 1. In (C)–(F), concentration
response curve for titan yellow is shown for comparison (dotted
lines). Raw data for all LOPAC samples can be found in Table S2.
8616 Nucleic Acids Research, 2012, Vol. 40, No. 17
Page 10
The interaction of HCV NS3 helicase with ssDNA was
monitored here with four different assays. The first, gel
shift analysis, is probably the most common method
used to study protein nucleic acid interactions. The gel
shift assay is laborious, not readily amenable to automa-
tion, and requires relatively large amounts of DNA and
protein. Of the three HTS compatible methods evaluated,
the FP-based method was the most precise, reproducible
and cost effective. FP-based assays have been used before
to measure the binding of helicases to a labeled substrate
(40,41), but have not been previously reported as methods
to screen for HCV helicase inhibitors. DNA binding to
NS3h can also be monitored either by measuring intrinsic
protein fluorescence (30,31) or by monitoring changes in
the fluorescence intensity of a fluorescein-labeled oligo-
nucleotide when it binds NS3h (9). Monitoring intrinsic
A
C
DE
B
Figure 6. Ability of compounds that disrupt the Cy5-dT15-NS3h complex to inhibit the HCV helicase and HCV replication. (A) Fluorescence
intensity of the MBHA substrate in assays containing increasing concentrations of suramin. (B) Effect of AG 538 (squares), ATA (circles), NF 023
(x), and suramin (triangles) on the initial rates of HCV helicase catalyzed DNA unwinding. Points are means of duplicate reactions, and error bars
are standard deviations. Data are fit to a normalized concentration response equation with parameters listed in Table 1. (C) The sub-genomic Renilla
luciferase reporter replicon used to monitor compound effects on HCV replication. (D) Relative HCV RNA levels in the presence of various
concentrations of AG 538 (squares), ATA (circles), NF 023 (x), and Suramin (triangles). Data are fit to a normalized concentration response
equation with parameters in Table 1. (E) Average (±SD) cell viability in assays where cells were exposed to 25 mM of indicated compounds.
ABC
Figure 7. Effect of compounds on the polarization of a Cy5-dT15-Escherichia coli single-stranded DNA binding protein (SSB) complex. (A)FP
based assay to monitor SSB binding to Cy5-dT15. (B) Binding between Cy5-dT15 and SSB determined by FP. (C) Cy-dT15-SSB complexes were
titrated with titan yellow (squares), ATA (diamonds), NF 023 (x), AG 538 (circles) or Suramin (inverted triangles). Assays were performed in
triplicate, points show means, and error bars standard deviations. Data are fit to a 4-parameter concentration equation constrained to values
obtained in the absence of inhibitor (top) and the absence of SSB (bottom).
Nucleic Acids Research, 2012, Vol. 40, No. 17 8617
Page 11
protein fluorescence is usually difficult in the presence of
small molecules, especially if they absorb light in the ultra-
violet wavelengths. Use of fluorescence intensity based
assays in screening is also difficult, because many library
samples fluoresce in the same range as fluorescein, and as
noted above, we have not yet found an alternate
red-shifted fluorophore that changes intensity when it
interacts with NS3h.
The LOPAC samples that most potently disrupted the
NS3h–DNA complex were two polysulfonated naphthy-
lureas (suramin and NF 023), a triphenylmethane (ATA)
and a tyrphostin (AG 538). All but the tyrphostin inhibit
helicase catalyzed strand separations. Tyrphostin AG 538
mimics a tyrosine kinase substrate so that it acts as a com-
petitive inhibitor of the IGF-1 receptor tyrosine kinase
(47). Preliminary mechanistic studies on each compound
suggest that only ATA inhibits the ability of NS3h to
cleave ATP in the absence of DNA or RNA, and all but
AG 538 prevent RNA from stimulating ATP hydrolysis.
Similarly, gel shift assays show all but AG 538 displace
NS3h from DNA, as was seen with thioflavine S
(Figure 1B). These data suggest that the AG 538
induced decrease in the polarization of both NS3h–
Cy5-dT15 and SSB–Cy5-dT15 complexes might be due
to a fluorescence artifact or something other than the
ability of the tyrphostin to prevent protein from binding
DNA. The ability of AG 538 to inhibit growth of the HCV
replicon is likewise probably not due to its effects on NS3.
AG 538 is an inhibitor of the insulin-like growth factor I
(IGF-1) tyrosine kinase, and it is possible IGF-1-mediated
signaling is needed for efficient HCV replication. Both
HCV infection and a reduction of IGF-1 levels are
linked to the development of liver cancer (48).
Compounds binding in place of ATP could also cause
the helicase to release its grip on DNA in the above
binding assay because ATP binding and hydrolysis
causes HCV helicase to cycle between low affinity and
high affinity DNA-binding states (28). When NS3h
releases DNA, it slides from the 3
0
to 5
0
end of a nucleic
acid as a Brownian motor (49). Thus, in light of the recent
demonstration that most nucleoside triphosphates can fuel
this motor action (6), it is not surprising that 2-methyl-
ATP, 2-(methylthio)ATP and 2-chloroATP were also hits
in the LOPAC screen. The fact that they inhibited binding
by only 30–40% of NS3h (which was less than our arbi-
trary 60% cutoff) is also not surprising since they were
tested at concentrations far less than the concentration of
ATP needed to fuel unwinding, or NS3 translocation, at
half-maximum rates (6). In contrast, a,b-methylene ATP
did not inhibit NS3h from binding dT15 (Table S1), con-
firming the earlier observations that most of the canonical
non-hydrolysable ATP analogs are poor inhibitors of
HCV helicase (22,50).
The fact that the polysulfonated naphthylureas and
triphenylmethanes affect HCV helicase is noteworthy
because the anti-microbial properties of these compounds
are well documented. Suramin has long been used to treat
sleeping sickness caused by trypanosomes, and it inhibits
protein tyrosine phosphatases (51) and G-proteins (52).
G-proteins and helicases share a similar Walker-type
nucleotide-binding site (52), and it is possible that suramin
inhibits HCV helicase and G-proteins through a similar
molecular mechanism. NF 023 is a suramin analog, P2X
receptor antagonist (53), and inhibitor of RNA editing in
trypanosomes (54). Suramin and NF 023 behave similarly
in all in vitro assays here, but it is noteworthy that (among
the two) only suramin was effective against the HCV
replicon in cells. A simple explanation would be that the
somewhat more aromatic suramin is more likely to enter
cells to exert an intracellular effect. Our results also
confirm the recently reported antiviral effect of ATA
against the HCV replicon, which had been reported after
ATA was found to inhibit the HCV NS5B RNA-
dependent RNA polymerase (55). ATA exerts a similar
Figure 8. Synthesis of specific HCV helicase inhibitors from a scaffold isolated from the yellow dye primuline. The flow chart summaries the source
of compounds tested (see text for details). The plot shows the ability of various primuline derivatives to inhibit HCV helicase (x-axis) and decrease
polarization of a Cy5-dT15-SSB complex. The dotted line shows where hypothetical compounds that inhibit both assays with the same potency
would lie on the plot. Structures of the four compounds with the most extreme properties are shown on the right, along with the ratios of IC
50
values
obtained in the two different assays. All data, structures and CID numbers can be found in Table S3.
8618 Nucleic Acids Research, 2012, Vol. 40, No. 17
Page 12
effect against a wide variety of enzymes that manipulate
nucleic acids, like human flap endonuclease 1 (FEN1) (56).
ATA also inhibits replication of influenzas A and B (57).
Triphenylmethanes that resemble ATA have been de-
veloped from the dye Soluble Blue HT as HCV helicase
inhibitors (14).
This study is focused on finding compounds that inhibit
nucleic acids from binding to the known, high
affinity-binding site on the NS3 helicase region. To best
target this site, we have used truncated NS3 lacking the
protease region (i.e. NS3h). Another advantage to using
NS3h instead of full length NS3 is that the truncated
protein expresses at higher levels in E. coli and is more
stable after purification. The same assays used here have
also been performed with full-length NS3 and NS3–NS4A
fusion peptides, with similar results, and it might be
possible to perform the screens here with both full-
length NS3 and NS3h to identify compounds that target
the still poorly defined nucleic acid binding sites on the
protease or in the cleft separating the protease from the
helicase. Such compounds might simultaneously inhibit
both NS3 protease and helicase activities. To date, no
small molecules that simultaneously inhibit both the
NS3 protease and helicase have been reported in the
academic literature, although a recent structure shows
that a protease inhibitor can interact with residues in
both the helicase and protease domains (58).
In any HTS campaign, it is important to have an
appropriate counter screen to identify non-specific com-
pounds. We show here that unrelated DNA binding
proteins can be substituted for NS3h in the FP-assay to
identify such compounds. The results show that the four
LOPAC hits affect both NS3h and E. coli SSB non-
specifically to inhibit the binding of the two proteins to
nucleic acids. As further evidence for a lack of specificity,
both suramin and ATA were hits in a LOPAC screen that
used a similar assay to identify compounds that prevent
the RNA-induced silencing complex from binding to
RNA (59). We show here how this SSB-based
counterscreen can be used to identify more specific NS3
helicase inhibitors using a library of recently disclosed
semi-synthetic analogs of potent helicase inhibitors
found in primuline (21). To identify primuline analogs
that have a greater affinity for HCV helicase than they
do for DNA, Li et al. (21) used an assay that monitors
the ability of the primuline derivatives to displace SYBR
Green I from DNA. As in this study, the most specific
derivative in the study by Li et al. was CID50930730
(Figure 7D), and the structure activity relationship
observed here with the SSB assay (Table S3) essentially
mirrors the relationships previously seen with DNA
binding data. The one important difference was that less
of each compound was needed to inhibit SSB binding than
was needed to displace SYBR Green I (21).
In conclusion, we established a new set of tools that can
be used to discover and analyze HCV helicase inhibitors.
None of these assays are new, and they have been exten-
sively reviewed elsewhere in the context of both DNA (60)
and RNA binding proteins (61). While the helicase inhibi-
tors identified from Sigma’s LOPAC are not specific for
NS3h, screens of larger, more diverse libraries might yield
potent, specific probes needed to study the role of HCV
helicase in cells. On the other hand, more specific analogs
of non-specific screening hits could be synthesized, as was
demonstrated above with the primuline derivatives.
Screens of the LOPAC and helicase inhibitor libraries
show that the binding assays presented here are easier to
interpret, and less prone to compound interference, than
assays monitoring helicase catalyzed DNA separation.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online:
Supplementary Tables 1–3.
ACKNOWLEDGEMENTS
We would like to thank Peter Hodder (Scripps Florida)
for valuable advice in assay development, and Seng-Lai
Tan for providing the HCV replicon.
FUNDING
National Institutes of Health [RO1 AI088001]; Research
Growth Initiative Award [101X219] from the University
of Wisconsin-Milwaukee Research Foundation; National
Institutes of Health Molecular Libraries Initiative [U54
HG005031]. Funding for open access charge: University
of Wisconsin-Milwaukee Research Foundation.
Conflict of interest statement. None declared.
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  • Source
    • "Understanding the mechanism of action of this molecular motor at the atomistic level is fundamental since the NS3 helicase domain has been proposed as a target for the development of a antiviral agents [6]. NS3 helicase has been characterized by single-molecule experiments7891011 and biochemical essays121314 . Although sometime acting as a dimer or as an oligomer [15, 16], NS3 also functions as a monomer, similarly to other SF1 and SF2 helicases [17, 18]. "
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    Full-text · Article · Aug 2015 · Nucleic Acids Research
  • Source
    • "Suramin appears to inhibit (re)initiation of CHIKV RNA synthesis, maybe by interfering with binding of the template RNA. This would be in line with earlier in vitro studies, reporting that suramin inhibits various RNA-binding enzymes like viral polymerases (De Clercq, 1979; Ono et al., 1988; Tarantino et al., 2014) and helicases (Basavannacharya and Vasudevan, 2014; Mukherjee et al., 2012). Several suramin-related compounds were analyzed, and though these compounds were not more effective, they provided insight into the (different) structural elements that are ND: EC 50 not determined due to low CC 50 . "
    [Show abstract] [Hide abstract] ABSTRACT: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe and often persistent arthritis. In recent years, millions of people have been infected with this virus for which registered antivirals are still lacking. Using our recently established in vitro assay, we discovered that the approved anti-parasitic drug suramin inhibits CHIKV RNA synthesis (IC50 of ∼5 μM). The compound inhibited replication of various CHIKV isolates in cell culture with an EC50 of ∼80 μM (CC50 >5 mM) and was also active against Sindbis virus and Semliki Forest virus. In vitro studies hinted that suramin interferes with (re)initiation of RNA synthesis, whereas time-of-addition studies suggested it to also interfere with a post-attachment early step in infection, possibly entry. CHIKV (nsP4) mutants resistant against favipiravir or ribavirin, which target the viral RNA polymerase, did not exhibit cross-resistance to suramin, suggesting a different mode of action. The assessment of the activity of a variety of suramin-related compounds in cell culture and the in vitro assay for RNA synthesis provided more insight into the moieties required for antiviral activity. The antiviral effect of suramin-containing liposomes was also analyzed. Its approved status makes it worthwhile to explore the use of suramin to prevent and/or treat CHIKV infections. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jun 2015 · Antiviral research
  • Source
    • "All but NF 023 inhibited replication of subgenomic HCV replicons (EC50 = 18–98 μM). Unfortunately, none of these inhibitors were specific to NS3 helicase [67]. "
    [Show abstract] [Hide abstract] ABSTRACT: Currently, hepatitis C virus (HCV) infection is considered a serious health-care problem all over the world. A good number of direct-acting antivirals (DAAs) against HCV infection are in clinical progress including NS3-4A protease inhibitors, RNA-dependent RNA polymerase inhibitors, and NS5A inhibitors as well as host targeted inhibitors. Two NS3-4A protease inhibitors (telaprevir and boceprevir) have been recently approved for the treatment of hepatitis C in combination with standard of care (pegylated interferon plus ribavirin). The new therapy has significantly improved sustained virologic response (SVR); however, the adverse effects associated with this therapy are still the main concern. In addition to the emergence of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase portion of the HCV NS3 protein. This review article summarizes our current understanding of HCV treatment, particularly with those of NS3 inhibitors.
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